Family Report for: PhoPQ_related

Autocrine proliferation repressor protein A (AprA) is a protein secreted by Dictyostelium discoideum cells. Although there is very little sequence similarity between AprA and any human protein, AprA has a predicted structural similarity to the human protein dipeptidyl peptidase IV (DPPIV). AprA is a chemorepellent for Dictyostelium cells, and DPPIV is a chemorepellent for neutrophils. This led us to investigate if AprA and DPPIV have additional functional similarities. We find that like AprA, DPPIV is a chemorepellent for, and inhibits the proliferation of, D. discoideum cells, and that AprA binds some DPPIV binding partners such as fibronectin. Conversely, rAprA has DPPIV-like protease activity. These results indicate a functional similarity between two eukaryotic chemorepellent proteins with very little sequence similarity, and emphasize the usefulness of using a predicted protein structure to search a protein structure database, in addition to searching for proteins with similar sequences.

Genetic screens based on the use of MudJ-generated lac fusions permitted the identification of novel genes regulated by the Rcs signal transduction system in Salmonella enterica serovar Typhimurium. Besides genes that are also found in the Escherichia coli genome, our screens identified Salmonella-specific genes regulated by RcsB, including bapA, siiE, srfA, and srfB. Here we show that the srfABC operon is negatively regulated by RcsB and by PhoP. In vivo studies using mutants with constitutive activation of the Rcs and/or PhoPQ system suggested that there is an overlap between these regulatory systems in the control of Salmonella virulence.

Many of the genes (pags (phoP activated genes) and prgs (phoP repressed genes)) regulated by the PhoP and PhoQ proteins (PhoP/Q) are necessary for survival of Salmonella typhimurium in murine macrophages and pathogenesis in mice. Although a great deal is known about the S. typhimurium phoP/Q regulon, little has been done with the human specific pathogen S. typhi, prompting us to investigate S. typhi phoP/Q regulated genes. Isogenic phoP12 (null) and phoP24 (constitutive) strains were constructed in S. typhi Ty2 and S. typhimurium C5 strains. Comparison of whole cell proteins from these strains by SDS-PAGE showed differences in both the number and molecular mass of PhoP/Q regulated proteins. This suggested that S. typhi and S. typhimurium may have different PhoP/Q regulated proteins and/or that their regulation may be different. A genetic procedure was developed to isolate mutations in PhoP/Q regulated genes. This involved random MudJ transposon mutagenesis of a phoP12 mutant, creating lacZ-gene fusions, and screening for Lac+ or Lac- colonies. A mobilizable plasmid carrying the phoP24 mutant gene was conjugated into these insertion mutants. Those that changed from Lac- to Lac+ were inferred to be pag::MudJ insertions and those that changed from Lac+ to Lac- were inferred to be prg::MudJ insertions. Five mutants with PhoP/Q regulated MudJ fusions were found by this scheme. The mutations were termed pqa (PhoPQ activated) and pqr (PhoPQ repressed) to distinguish them from other PhoP/Q regulated genes. The pqa/pqr::MudJ mutations were transduced into S. typhi phoP+ and phoP24 strains by Vi-l phage transduction. Characterization of the mutants (Southern blot analysis, beta-galactosidase activity on indicator plates and in liquid cultures) strongly suggested that their MudJ insertion mutations were in five different genes. Further characterization involved determining cationic peptide sensitivity and mouse virulence. Two mutants were found to be sensitive to the antimicrobial peptide melittin.

Autocrine proliferation repressor protein A (AprA) is a protein secreted by Dictyostelium discoideum cells. Although there is very little sequence similarity between AprA and any human protein, AprA has a predicted structural similarity to the human protein dipeptidyl peptidase IV (DPPIV). AprA is a chemorepellent for Dictyostelium cells, and DPPIV is a chemorepellent for neutrophils. This led us to investigate if AprA and DPPIV have additional functional similarities. We find that like AprA, DPPIV is a chemorepellent for, and inhibits the proliferation of, D. discoideum cells, and that AprA binds some DPPIV binding partners such as fibronectin. Conversely, rAprA has DPPIV-like protease activity. These results indicate a functional similarity between two eukaryotic chemorepellent proteins with very little sequence similarity, and emphasize the usefulness of using a predicted protein structure to search a protein structure database, in addition to searching for proteins with similar sequences.

BACKGROUND: Dictyostelium cells secrete the proteins AprA and CfaD. Cells lacking either AprA or CfaD proliferate faster than wild type, while AprA or CfaD overexpressor cells proliferate slowly, indicating that AprA and CfaD are autocrine factors that repress proliferation. CfaD interacts with AprA and requires the presence of AprA to slow proliferation. To determine if CfaD is necessary for the ability of AprA to slow proliferation, whether AprA binds to cells, and if so whether the binding requires the presence of CfaD, we examined the binding and effect on proliferation of recombinant AprA.
RESULTS: We find that the extracellular accumulation of AprA increases with cell density and reaches a concentration of 0.3 microg/ml near a stationary cell density. When added to wild-type or aprA- cells, recombinant AprA (rAprA) significantly slows proliferation at 0.1 microg/ml and higher concentrations. From 4 to 64 microg/ml, the effect of rAprA is at a plateau, slowing but not stopping proliferation. The proliferation-inhibiting activity of rAprA is roughly the same as that of native AprA in conditioned growth medium. Proliferating aprA- cells show saturable binding of rAprA to 92,000 +/- 11,000 cell-surface receptors with a KD of 0.03 +/- 0.02 microg/ml. There appears to be one class of binding site, and no apparent cooperativity. Native AprA inhibits the binding of rAprA to aprA- cells with a Ki of 0.03 mug/ml, suggesting that the binding kinetics of rAprA are similar to those of native AprA. The proliferation of cells lacking CrlA, a cAMP receptor-like protein, or cells lacking CfaD are not affected by rAprA. Surprisingly, both cell types still bind rAprA. CONCLUSION: Together, the data suggest that AprA functions as an autocrine proliferation-inhibiting factor by binding to cell surface receptors. Although AprA requires CfaD for activity, it does not require CfaD to bind to cells, suggesting the possibility that cells have an AprA receptor and a CfaD receptor, and activation of both receptors is required to slow proliferation. We previously found that crlA- cells are sensitive to CfaD. Combined with the results presented here, this suggests that CrlA is not the AprA or CfaD receptor, and may be the receptor for an unknown third factor that is required for AprA and CfaD activity.

Dictyostelium discoideum cells secrete CfaD, a protein that is similar to cathepsin proteases. Cells that lack cfaD proliferate faster and reach a higher stationary-phase density than wild-type cells, whereas cells that overexpress CfaD proliferate slowly and reach the stationary phase when at a low density. On a per-nucleus basis, CfaD affects proliferation but not growth. The drawback of not having CfaD is a reduced spore viability. Recombinant CfaD has no detectable protease activity but, when added to cells, inhibits the proliferation of wild-type and cfaD(-) cells. The secreted protein AprA also inhibits proliferation. AprA is necessary for the effect of CfaD on proliferation. Molecular-sieve chromatography indicates that in conditioned growth medium, the 60 kDa CfaD is part of a approximately 150 kDa complex, and both chromatography and pull-down assays suggest that CfaD interacts with AprA. These results suggest that two interacting proteins may function together as a chalone signal in a negative feedback loop that slows Dictyostelium cell proliferation.

Genetic screens based on the use of MudJ-generated lac fusions permitted the identification of novel genes regulated by the Rcs signal transduction system in Salmonella enterica serovar Typhimurium. Besides genes that are also found in the Escherichia coli genome, our screens identified Salmonella-specific genes regulated by RcsB, including bapA, siiE, srfA, and srfB. Here we show that the srfABC operon is negatively regulated by RcsB and by PhoP. In vivo studies using mutants with constitutive activation of the Rcs and/or PhoPQ system suggested that there is an overlap between these regulatory systems in the control of Salmonella virulence.

Many cells appear to secrete factors called chalones that limit their proliferation, but in most cases the factors have not been identified. We found that growing Dictyostelium cells secrete a 60 kDa protein called AprA for autocrine proliferation repressor. AprA has similarity to putative bacterial proteins of unknown function. Compared with wild-type cells, aprA-null cells proliferate faster, while AprA overexpressing cells proliferate slower. Growing wild-type cells secrete a factor that inhibits the proliferation of wild-type and aprA- cells; this activity is not secreted by aprA- cells. AprA purified by immunoprecipitation also slows the proliferation of wild-type and aprA- cells. Compared with wild type, there is a higher percentage of multinucleate cells in the aprA- population, and when starved, aprA- cells form abnormal structures that contain fewer spores. AprA may thus decrease the number of multinucleate cells and increase spore production. Together, the data suggest that AprA functions as part of a Dictyostelium chalone.

Many of the genes (pags (phoP activated genes) and prgs (phoP repressed genes)) regulated by the PhoP and PhoQ proteins (PhoP/Q) are necessary for survival of Salmonella typhimurium in murine macrophages and pathogenesis in mice. Although a great deal is known about the S. typhimurium phoP/Q regulon, little has been done with the human specific pathogen S. typhi, prompting us to investigate S. typhi phoP/Q regulated genes. Isogenic phoP12 (null) and phoP24 (constitutive) strains were constructed in S. typhi Ty2 and S. typhimurium C5 strains. Comparison of whole cell proteins from these strains by SDS-PAGE showed differences in both the number and molecular mass of PhoP/Q regulated proteins. This suggested that S. typhi and S. typhimurium may have different PhoP/Q regulated proteins and/or that their regulation may be different. A genetic procedure was developed to isolate mutations in PhoP/Q regulated genes. This involved random MudJ transposon mutagenesis of a phoP12 mutant, creating lacZ-gene fusions, and screening for Lac+ or Lac- colonies. A mobilizable plasmid carrying the phoP24 mutant gene was conjugated into these insertion mutants. Those that changed from Lac- to Lac+ were inferred to be pag::MudJ insertions and those that changed from Lac+ to Lac- were inferred to be prg::MudJ insertions. Five mutants with PhoP/Q regulated MudJ fusions were found by this scheme. The mutations were termed pqa (PhoPQ activated) and pqr (PhoPQ repressed) to distinguish them from other PhoP/Q regulated genes. The pqa/pqr::MudJ mutations were transduced into S. typhi phoP+ and phoP24 strains by Vi-l phage transduction. Characterization of the mutants (Southern blot analysis, beta-galactosidase activity on indicator plates and in liquid cultures) strongly suggested that their MudJ insertion mutations were in five different genes. Further characterization involved determining cationic peptide sensitivity and mouse virulence. Two mutants were found to be sensitive to the antimicrobial peptide melittin.